Glucose 6-phosphatase activity in pregnant and lactating mammary glands of the mouse.код для вставкиСкачать
THE ANATOMICAL RECORD 214:383-388 (1986) Glucose 6-Phosphatase Activity in Pregnant and Lactating Mammary Glands of the Mouse YOSHIHIKO SHUGYO, J U N WATANABE, SHINSUKE KANAMURA, AND KAZUO KANA1 Department of Anatomy, Kansai Medical University, Moriguchi Osaka, 570 Japan ABSTRACT Glucose 6-phosphatase activity was studied in the secretory epithelial cell and other cell types composing alveoli of the mammary gland (cytochemical study) and in the whole mammary gland (biochemical study) of pregnant and lactating mice. The reaction product for the enzyme activity was seen in the endoplasmic reticulum and nuclear envelope in secretory epithelial cells from all animals examined (days 7 and 14 of pregnancy, and days 0, 3, 10, and 20 of lactation. The amounts of the reaction product appeared scarce a t day 7 of pregnancy, moderate at day 14 of pregnancy and day 0 of lactation, and abundant at days 3 and 10 of lactation. The reaction product, however, became generally scarce at day 20 of lactation. Biochemical activity was relatively low a t days 7 and 14 of pregnancy and days 0 and 20 of lactation, while it was high a t days 3 and 10 of lactation. The increased activity is probably related to functions of secretory epithelial cells in the lactating gland. Glucose 6-phosphatase (GGPase; EC184.108.40.206, D-glucose 6-phosphate phosphohydrolase) activity is shown to be generally high in functionally active cells, in which the endoplasmic reticulum is well developed, such as the hepatocyte (Tice and Barrnett, 1962; Ericsson, 1966; Kanamura, 1971a,b, 197513; Leskes et al., 1971) and jejunal epithelial cell (Hugon et al., 1970,1971).The secretory epithelial cell in pregnant and lactating mammary glands is also a functionally active cell bearing welldeveloped endoplasmic reticulum and numerous secretory granules. On the other hand, glucose is present in milk (Reineccius et al., 1970; Baldwin and Lang, 1974; Kuhn and White, 1975). G6Pase is concerned with glucose release into tissue fluids, such as the blood (Nordlie, 1972) and epididymal fluid (Kanai et al., 1981, 1983),by hydrolysis of glucose 6-phosphate (G6P). It is therefore of interest to examine whether G6Pase activity is present in the secretory epithelial cell of the pregnant and lactating mammary glands. However, the presence of G6Pase activity in the secretory cell of the mammary gland has not been demonstrated, although there is a report about hydrolysis of glucose 6-phosphate by homogenates of rat mammary gland (Threadgold and Kuhn, 1979). In the present paper, we describe G6Pase activity in the secretory epithelial cell and other cell types composing alveoli of the mammary gland (cytochemical study) and in the whole mammary gland (biochemical study) of pregnant and lactating mice, and discuss possible roles of the enzyme in the mammary gland. male for one night; the following day was designated day 1of pregnancy. Lactating mice were used from the time they gave birth to their litter but before nursing (designated day 0 of lactation). Animals were examined a t days 7 and 14 of pregnancy, and days 0 , 3 , 10, and 20 of lactation. The animals had free access to food and water prior to the experiments. Cytochemical methods Left inguinal mammary glands of the animals under Nembutal anaesthesia were perfused via the left ventricle, first with 0.9% saline for about 30 seconds, and then with 2% glutaraldehyde containing 0.1 M sodium cacodylate (pH 7.2) for 5 minutes (3 ml/minute). The tissues were washed briefly in 0.1 M sodium cacodylate (pH 6.7) containing 8% sucrose a t 4°C (Kanamura, 19731, sectioned a t 40 pm by a freezing microtome, and incubated in a reaction medium (3.7 mM G6P, 80 mM sodium cacodylate, 3.6 mM lead nitrate, and 230 mM sucrose, pH 6.7; Watanabe et al., 1983; Kanai et al., 1983)for 60 minutes at room temperature with a change of the medium. The sections were postfixed in 1%buffered osmium tetroxide (pH 7.4) a t 4°C for 3 hours, dehydrated, and embedded in Spurr. Thin sections were cut with glass knives on a n LKB ultrotome, stained with uranyl acetate and lead citrate, and examined in a JEM 100-5 electron microscope. Observations were carried out on alveoli of the mammary gland. For light microscopic observations, 40-pm-thick sections incubated in the reaction medium were washed MATERIALS AND METHODS Female ddY mice about 3 months old were used. Pregnant mice were obtained by mating female mice with 0 1986 ALAN R. LISS, INC. Received August 6, 1985; accepted October 30,1985. 384 Y. SHUGYO, J. WATANABE, S. KANAMURA. AND K. KANA1 with the buffer, immersed briefly in ammonium sulfide, washed again, and mounted in glycerine jelly. In order to ascertain whether the reaction product is due to G6Pase activity, control experiments were carried out (Kanamura, 1971a). The experiments consisted of incubation of the fixed sections in the reaction medium lacking G6P; or incubation in 0.1 M acetate buffer (pH 5.0) a t 37°C for 15 minutes before incubation in the reaction medium; or incubation in a reaction medium containing equal moles of 0-glycerophosphate in place of G6P; or pre-incubation in 0.25 M sucrose containing 10 mM NaF for 15 minutes and then incubation in the reaction medium containing equal moles of NaF. Biochemical methods Five animals were used per each day group of pregnancy or lactation. Mammary glands were homogenized at 4°C in 0.25 M sucrose (2%w/v) in a Potter-Elvehjem homogenizer for 5 minutes a t 4,000 redminute. The homogenates were centrifuged at 3,000 redminute for 10 minutes at 4°C and assayed for G6Pase activity according to the method described by Leskes et al. (1971). Levamisole (10 mM) was added to the reaction medium in order to inhibit alkaline phosphatase activity. Incubation time was 30 minutes. The inorganic phosphorus released was determined by the method of Fiske and SubbaRow (1925). Enzyme activity was expressed as pg phosphorus liberatediminutelmg wet tissue. RESULTS Cytochemical results In secretory epithelial cells from all animals examined (days 7 and 14 of pregnancy and days 0, 3, 10, and 20 of lactation), the reaction product for G6Pase activity was seen in the endoplasmic reticulum and nuclear envelope (Figs. 1-7). The amounts of the reaction product appeared scarce at day 7 of pregnancy (Fig. 11, moderate a t day 14 of pregnancy and day 0 of lactation (Figs. 2, 31, and abundant a t days 3 and 10 of lactation (Figs. 4, 6a). The reaction product, however, became scarce or sometimes moderate at day 20 of lactation (Fig. 7). The deposition of final product was occasionally found also in lysosomes. Mitochondria, Golgi apparatus, dense-protein granules, and plasma membrane showed no reaction product. Figs. 1-7.Cytochemical demonstration of glucose 6-phosphatase ac- product is abundant in the endoplasmic reticulum and nuclear envetivity in the cells of alveoli of mammary glands from pregnant and lope. x 13,000. lactating mice. Sections (30 pm) cut from perfusion-fixed tissues were Fig. 5. Secretory epithelial cell and myoepithelial cell at day 3 of incubated in a medium modified from that of Wachstein and Meisel lactation. Fixed sections were immersed at 37°C in 0.1 M acetate for 60 minutes. buffer and then incubated in the reaction medium. The reaction prodFig. 1. Secretory epithelial cells at day 7 of pregnancy. The reaction uct is absent from the endoplasmic reticulum and nuclear envelope of product (arrowheads) is scarce in the endoplasmic reticulum and nu- secretory epithelial cell, but abundant in the plasma membrane of myoepithelial cell. x 10,000, clear envelope. x 15,000. Figs. 2,3.Secretory epithelial cells at day 14 of pregnancy and day 0 of lactation, respectively. Moderate amount of the reaction product is seen in the endoplasmic reticulum and nuclear envelope. x 10,000. Fig. 4. Secretory epithelial cells at day 3 of lactation. The reaction Fig. 6.Secretory epithelial cells (Fig. 6a, x 15,000)and myoepithelial cell (Fig. 6b, ~ 8 , 0 0 0at ) day 10 of lactation. In the secretory cells, the reaction product is abundant in the endoplasmic reticulum and nuclear envelope. The reaction product is also seen in the endoplasmic reticulum and nuclear envelope of myoepithelial cell. G6PASE IN MAMMARY GLAND 385 386 Y. SHUGYO, J. WATANABE, S. KANAMURA, AND K. KANA1 between day 7 of pregnancy and day 3 of lactation ( P < .Ol), and between days 10 and 20 of lactation (P < .05) were significant. DISCUSSION Fig. 7. Secretory epithelial cells at day 20 of lactation. The reaction product is (a) scarce or (b)moderate in the endoplasmic reticulum and nuclear envelope. x 13,000. The reaction product for G6Pase activity was also present in the endoplasmic reticulum and nuclear envelope in other cell types composing alveoli of pregnant and lactating mammary glands, i.e., myoepithelial cells (Fig. 6b), fibroblasts, and endothelial cells of capillaries. The amount of the reaction product was moderate or abundant in myoepithelial cells, but scarce in the other two cell types. A scarce amount of final product was sometimes observed also in the plasma membrane of myoepithelial cells, fibroblasts, and endothelial cells. Mitochondria and Golgi apparatus in these cells showed no reaction product. Omission of G6P from the incubation medium resulted in a complete absence of the reaction product. Immersion of the fixed sections in 0.1 M acetate buffer (pH 5.0) before incubation in the reaction medium or use of 0glycerophosphate in place of G6P in the reaction medium caused a loss of the reaction product except in lysosomes and the plasma membrane. Pre-incubation of the fixed sections in 0.25 M sucrose containing 10 mM NaF' for 15 minutes, followed by incubation in the reaction medium containing equal moles of NaF, abolished the total reaction, but the final product was still visible on the plasma membrane. These results indicate that the reaction product in the endoplasmic reticulum and nuclear envelope is due to G6Pase activity, but the deposition of final product in the plasma membrane of myoepithelial cells, fibroblast, and endothelial cells or lysosomes in secretory epithelial cells is probably related to nonspecific phosphatase or acid phosphatase activity. As revealed in the present cytochemical results, G6Pase activity in the endoplasmic reticulum and nuclear envelope of secretory epithelial cells is higher in mammary glands of days 3 and 10 of lactation than in pregnant and weaning glands. The biochemical activity was relatively low at days 7 and 14 of pregnancy, and days 0 and 20 of lactation, while it was high at days 3 and 10 of lactation. Thus, the biochemical results paralleled generally those of cytochemical experiments. The higher G6Pase activity is probably related to functions of secretory epithelial cells of the lactating gland. G6Pase has a wide spectrum of hydrolytic and synthetic activities (Nordlie, 1972). However, hydrolysis of G6P is probably the sole function of this enzyme in vivo (Arion et al., 1972).The activity is high in hepatocytes, proximal convoluted tubule cells of the kidney, and jejunal epithelial cells. The functional role of the enzyme in the liver and kidney is to release glucose into blood by hydrolyzing G6P produced via gluconeogenesis and glycogenolysis (Krebs, 1963; Nordlie, 1972).The enzyme in the jejunal epithelium might be concerned with the absorption of nutrients. We recently observed relatively high G6Pase activity in principal cells of mouse ductus epididymidis and postulated that the role of the enzyme is to supply glucose into the epididymal fluid for use by spermatozoa (Kanai et al., 1981,1983). However, the role of this enzyme in other cell types containing low or moderate activity is unknown, although we supposed a role of regulation of G6P concentration in the cells, h I 7 14 0 3 10 20 Pregnancy (days) Lpatm Biochemical results Fig. 8. G6Pase activity in pregnant and lactating mammary glands The activity (pg phosphorus130 minuteslmg wet tissue) of mice. Each point represents the mean of five animals and standard was relatively low a t days 7 and 14 of pregnancy and error is shown as a vertical line. Differences in the values between day days 0 and 20 of lactation, while it was high at days 3 7 of pregnancy and day 3 of lactation ( P < ,011, and between days 10 and 10 of lactation (Fig. 8). Differences in the values and 20 of lactation ( P < .05) are significant. 387 GGPASE IN MAMMARY GLAND hydrolyzing any excess (Kanamura, 1975a; Watanabe et al.. 1983. 1986). One role of G6Pase in secretory epithelial cells of the lactating mammary gland may be also to regulate the concentration of G6P in the cells. The lactating mammary gland consumes a large amount of glucose (Linzell, 1968; Lindsay, 1971). Hexokinase that synthesizes G6P from blood glucose is shown to be high in the lactating mammary gland (Walter and McLean, 1967). G6Pase probably hydrolyzes G6P that is steadily produced by hexokinase, if there is any excess. In addition to lactose, the milk generally contains a number of other carbohydrates. Glucose and galactose are usually detected in low concentrations. Having taken precautions to avoid hydrolysis of lactose, Reineccius et al. (1970)found 0.77 mM glucose in cow’s milk. Baldwin and Lang (1974) and Kuhn and White (1975) found 0.29 mM and 0.5-1 mM glucose in rat milk. However, we failed to find in the literature the data of glucose in mouse milk. Although very pronounced species differences are evident in the proportions of individual carbohydrates, it is probably impossible that certain carbohydrates in the milk occur in one species but are completely absent from another (Baldwin and Lang, 1974). Thus, mouse milk probably contains glucose. It is therefore likely that one role of G6Pase in secretory epithelial cells of the lactating mammary gland is to release glucose into the milk by hydrolizing G6P. Various hormones, such as estrogens, progesterone, prolactin, glucocorticoids, growth hormone, thyroid hormones, insulin, and placental lactogen influence the mammary development and lactation. In the mouse, plasma concentrations of prolactin and progesterone, after showing a peak during the first half of pregnancy, increase toward the end of pregnancy and then fall on the day of parturition (Murr et al., 1974; Virgo and Bellward, 1974). Prolactin levels are high during lactation in many species (Topper and Freeman, 1980). Plasma estradiol-170 increases close to parturition (McCormack and Greenwald, 1974). Serum levels of corticosterone are high a t the end of pregnancy and decrease during parturition and early lactation (Gala and Westphal, 1967). From the present results, it is not clear which hormones are related to the changes in G6Pase activity. Most enzyme levels in mouse mammary gland cells are affected by prolactin, glucocorticoids, and thyroid hormones (Banerjee, 1976; Topper and Freeman, 1980).Prolactin and thyroid hormones are suggested to regulate the rate of synthesis of the mammary enzymes by modifying nuclear RNA synthesis (Banerjee, 1976). Prolactin, glucocorticoids, and thyroid hormones might relate to regulation of G6Pase synthesis. G6Pase activity is present in a variety of cell types of various organs, such as hepatocytes (Kanamura, 1971a,b, 1975b; Leskes et al., 1971),proximal convoluted tubule cells of the kidney (Kanamura, 1971c), jejunal epithelial cells (Hugon et al., 1970, 1971), pancreatic B cells (Lazarus and Barden, 19651, follicular epithelial cells of the thyroid gland (Rosen, 19721, cells of the epididymis (Kanai et al., 1981, 19831, and submandibular acinar cells (Watanabe et al., 1983). In all these cell types as well as the secretory epithelial cells in the mammary gland, as revealed in the present results, G6Pase activity is exclusively localized in the endoplasmic reticulum and nuclear envelope. Threadgold and Kuhn (1979) reported that G6P hydrolysis by preparations of lactating rat mammary gland was not due to G6Pase and could not be utilized as a microsomal marker. However, the reaction medium used for hydrolysis of G6P contained no inhibitor for alkaline or acid phosphatase. The authors stated as reasons for denying G6Pase activity that G6P a s well as a wide range of other phosphates were hydrolyzed by the mammary gland preparation, and that the hydrolysis of G6P was maximal a t pH 5 and 10 or above, and minimal a t pH 6.4-7. The mammary gland preparation probably includes alkaline and acid phosphatase activities, and, therefore, such preparation hydrolyzes various phosphates. It is likely that peaks of hydrolysis at pH 5 and 10 or above are by acid and alkaline phosphatase activities, and the minimal activity at pH 6.4-7 is G6Pase activity. As revealed in the present study, the exclusive localization in the endoplasmic reticulum and nuclear envelope and results of control experiments ensure the presence of G6Pase activity in secretory epithelial cells of the mouse mammary gland. LITERATURE CITED Arion, W.J., B.K. Wallin, P.W. Carlson, and A.J. Lange (1972) The specificity of glucose 6-phosphatase of intact liver microsomes. J. Biol. 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